Evaluation on purine metabolism in human skin fibroblast cells exposed to oxygenated polycyclic aromatic hydrocarbons.

A rapid and sensitive assessment of the toxicity of oxygenated polycyclic aromatic hydrocarbons (OPAHs), widely distributed persistent organic pollutants in the environment, is crucial for human health. In this study, using high-performance liquid chromatography, the separation and detection of four purines, xanthine (X), guanine (G), adenine (A), and hypoxanthine (HX) in cells were performed. The aim was to evaluate the cytotoxicity of three OPAHs, namely 1,4-benzoquinone (1,4-BQ), 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone (9,10-PQ), with higher environmental concentrations, from the perspective of purine nucleotide metabolism in human skin fibroblast cells (HFF-1). The results revealed that the levels of G and A were low in HFF-1 cells, while the levels of HX and X showed a dose-response relationship with persistent organic pollutants concentration. With increased concentration of the three persistent organic pollutants, the purine metabolism in HFF-1 cells weakened, and the impact of the three persistent organic pollutants on purine metabolism in cells was in the order of 9,10-PQ > 1,4-BQ > 1,2-NQ. This study provided valuable insights into the toxic mechanisms of 1,4-BQ, 1,2-NQ and 9,10-PQ, contributing to the formulation of relevant protective measures and the safeguarding of human health.

Hydrophilic interaction liquid chromatography based method for simultaneous determination of purines and their derivatives in food spices.

This work presents method for separation and quantification of adenine, guanine, xanthine, hypoxanthine, uric acid, and creatinine in food spices using hydrophilic interaction liquid chromatography with UV detection. Optimized conditions allowed separation with mobile phases containing acetonitrile and additives ammonium acetate (90:10, v/v, pH 6.1) or formate (90:10, v/v, pH 3.2). In food spices no uric acid was detected, creatinine (16 ± 2 µg g-1) was found only in instant dried yeast. The highest content of purines was determined in dried yeast (xanthine 110 ± 8 µg g-1, hypoxanthine 441 ± 24 µg g-1, adenine 84 ± 16 µg g-1, guanine 163 ± 12 µg g-1), high in curry, herbal pepper, and chicken seasoning, the lowest concentration was in black pepper (hypoxanthine 12 ± 2 µg g-1, adenine 27 ± 3 µg g-1). To best of our knowledge, no such complementary method and obtained data have been reported so far.

Determination of purines in prepackaged food using optimum acid hydrolysis followed by high performance liquid chromatography.

A high performance liquid chromatography was established to determine purine content of prepackaged food. Chromatographic separation was performed on Agilent5 TC-C18 column. Ammonium formate (10 mmol/L, pH = 3.385) and methanol (99:1) were used as mobile phase. Purine concentration and peak area showed good linear relationships in the range from 1 to 40 mg/L (guanine, hypoxanthine, adenine) and xanthine exhibited a good linear relationship ranged from 0.1 to 4.0 mg/L. Recoveries of four purines ranged from 93.03% to 107.42%. Purine content in prepackaged food was following: animal derived prepackaged food: 16.13-90.18 mg/100 g; beans and bean products: 66.36-157.11 mg/100 g; fruits and fruit products: 5.64-21.79 mg/100 g; instant rice and flour products: 5.68-30.83 mg/100 g; fungi, algae, fungi and algae products: 32.57-70.59 mg/100 g. This proposed method had good precision and accuracy with a wide linear range for detection of purine. Animal derived prepackaged food was purine-rich food, purine content of plant derived prepackaged food varied greatly.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Altered Follicular Fluid Metabolic Pattern Correlates with Female Infertility and Outcome Measures of In Vitro Fertilization.

Nearly 40-50% of infertility problems are estimated to be of female origin. Previous studies dedicated to the analysis of metabolites in follicular fluid (FF) produced contrasting results, although some valuable indexes capable to discriminate control groups (CTRL) from infertile females (IF) and correlate with outcome measures of assisted reproduction techniques were in some instances found. In this study, we analyzed in blind FF of 35 control subjects (CTRL = patients in which inability to obtain pregnancy was exclusively due to a male factor) and 145 IF (affected by: endometriosis, n = 19; polycystic ovary syndrome, n = 14; age-related reduced ovarian reserve, n = 58; reduced ovarian reserve, n = 29; unexplained infertility, n = 14; genetic infertility, n = 11) to determine concentrations of 55 water- and fat-soluble low molecular weight compounds (antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines, energy-related metabolites, and amino acids). Results evidenced that 27/55 of them had significantly different values in IF with respect to those measured in CTRL. The metabolic pattern of these potential biomarkers of infertility was cumulated (in both CTRL and IF) into a Biomarker Score index (incorporating the metabolic anomalies of FF), that fully discriminated CTRL (mean Biomarker Score value = 4.00 ± 2.30) from IF (mean Biomarker Score value = 14.88 ± 3.09, p < 0.001). The Biomarker Score values were significantly higher than those of CTRL in each of the six subgroups of IF. Posterior probability curves and ROC curve indicated that values of the Biomarker Score clustered CTRL and IF into two distinct groups, based on the individual FF metabolic profile. Furthermore, Biomarker Score values correlated with outcome measures of ovarian stimulation, in vitro fertilization, number and quality of blastocysts, clinical pregnancy, and healthy offspring. These results strongly suggest that the biochemical quality of FF deeply influences not only the effectiveness of IVF procedures but also the following embryonic development up to healthy newborns. The targeted metabolomic analysis of FF (using empowered Redox Energy Test) and the subsequent calculation of the Biomarker Score evidenced a set of 27 low molecular weight infertility biomarkers potentially useful in the laboratory managing of female infertility and to predict the success of assisted reproduction techniques.

[Separation and identification of impurities from intermediates of istradefylline].

Istradefylline is a novel selective adenosine A2A receptor antagonist that is used to treat Parkinson's disease and improve motor dysfunction in the early stage of this disease. During the synthesis of intermediate A1 (6-amino-1,3-diethyl-2,4-(1H,3H)-pyrimidinedione), at least two by-products were formed under alkaline or high-temperature conditions. In a previous study, one of the by-products in the synthesis of the intermediate was studied, and its structure was identified as (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide. In this study, we used high performance liquid chromatography (HPLC) to analyze another impurity formed during the synthesis of A1, and the following steps were executed: 0.4 g of intermediate was weighed and added to a 50 mL beaker, followed by the sequential addition of 8 mL water and 8 mL acetonitrile, and then, ultrasonic dissolution was performed. Finally, the solution was filtered through a 0.45-µm organic membrane and the test sample solution was obtained. We used the Agilent zorbax C18 chromatography column, with acetonitrile (A)/water(B) as the mobile phase under gradient elution ((tmin/A∶B)=t0/20∶80, t15/60∶40, t20-t50/90∶10). The detector wavelength is 268 nm. In order to separate the impurity from A1, we used a Ceres B preparative column, with acetonitrile-water (30/70, v/v) as the mobile phase. The flow rate was set at 30 mL/min, and the detection wavelength was 268 nm. The structure of the impurity was confirmed by high-resolution mass spectrometry (HRMS), one-dimensional nuclear magnetic resonance (NMR), and two-dimensional nuclear magnetic resonance (2D NMR), and characterized by single-crystal X-ray diffraction (XRD). In MS experiments, an electrospray ionization (ESI) source was used with positive ion scanning. In the NMR experiments, we used tetramethylsilane (TMS) as the internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as the solvent to obtain the spectra. The results of preparative high performance liquid chromatography (Prep-HPLC) showed that good separation effect could be achieved by isocratic elution, and the impurity was perfectly separated. The1H-NMR spectral data are as follows:1H-NMR (600 MHz, DMSO): δ 1.01 (q, J=6.9 Hz, 3H), 1.02 (q, J=6.9 Hz, 3H), 1.07 (t, J=6.9 Hz, 3H), 3.04 (p, J=6.8 Hz, 2H), 3.74 (q, J=7.0 Hz, 2H), 3.94 (q, J=7.1 Hz, 2H), 5.85 (s, 1H). The 13C-NMR spectral data are summarized as follows: 13C-NMR (150 MHz, DMSO): δ13.9, 14.1, 15.9, 34.6, 34.9, 36.9, 81.9, 152.2, 153.3, 159.3, 162.0. The impurity was characterized by single-crystal XRD, and its spatial structure was further verified and determined as 1-(1,3-diethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-3-ethylurea. Based on the chemical structure of the impurity, we propose the following mechanism for the impurity: when A1 is synthesized under alkaline conditions or at high temperature, excessive diethylurea continues to undergo amidation with A1 to obtain this by-product. Although the formation mechanism of the impurity studied in this paper is different from that of the intermediate A1 impurity (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide, both the impurities are formed at high temperatures. Both will be accompanied by A1 in the subsequent reaction of istradefylline synthesis. The relationship between drug impurities and drug safety is a complex relationship that is affected by many factors. Generally, most impurities in drugs have potential biological activities, and some even interact with the drugs, thus affecting their efficacy and safety and inducing toxic effects. Therefore, to ensure the quality of istradefylline, it is necessary to control the impurity content during the production. The findings of this paper may provide guidelines for controlling the impurity content in istradefylline.

A Highly Sensitive Nonextraction-Assisted HPLC Method with Fluorescence Detection for Quantification of Duvelisib in Plasma Samples and its Application to Pharmacokinetic Study in Rats.

BACKGROUND: Duvelisib (DUV) is a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor. It has been recently granted an accelerated approval for treatment of adult patients with relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). It is also effective in therapy of T-cell lymphoma, solid tumors, and non-Hodgkin's lymphoma. In literature, there is no method valid for quantitation of DUV in human plasma for its therapeutic monitoring and pharmacokinetic studies. PURPOSE: The purpose of this study is the establishment of a highly sensitive HPLC method with fluorescence detection for quantitation of DUV in plasma for its therapeutic monitoring and pharmacokinetic studies of DUV. METHODS: The resolution of DUV and the internal standard (IS) olaparib (OLA) was achieved on Nucleosil CN column, with a mobile phase composed of acetonitrile:water (25:75, v/v) at a flow rate of 1.7 mL min-1. The fluorescence of both DUV and OLA was detected at 410 nm after excitation at 280 nm. The method was validated according to the guidelines of bioanalytical method validation. RESULTS: The method was linear in the range of 5-100 ng mL-1, and its limit of detection (LOD) and limit of quantitation (LOQ) were 2.12 ng mL-1 and 7 ng mL-1, respectively. The precisions of the method were ≤ 8.26%, and its accuracies were ≥ 95.32%. All the other validation parameters were satisfactory. The proposed method was successfully employed to the investigation of the pharmacokinetic profile of DUV in rats following a 25 mg/kg single dose of oral administration. CONCLUSION: The method is characterized with high sensitivity, accuracy, simple sample pretreatment, rapidity, eco-friendly as it consumes low volumes of organic solvent in the mobile phase and has high analysis throughput as its run time was short (~ 10 min).

Meat quality and Raman spectroscopic characterization of Korat hybrid chicken obtained from various rearing periods.

Meat quality attributes vary with chicken age. Understanding the relationship between poultry age and the quality of the meat would be beneficial for efficient poultry farming to meet market needs. The Korat hybrid chicken (KC) is a new crossbred chicken whose meat quality is distinct from that of commercial broiler (CB) chickens and has not been well characterized. In this study, we characterized the physico-chemical properties of KC meat and correlate the findings with Raman spectral data. The protein content of KC breast and thigh meat increased with age. The pH of thigh meat decreased, while the water-holding capacity of breast meat increased as the age of the chickens increased. The amount of cholesterol in breast meat decreased as the rearing period was extended. Inosine 5'-monophosphate and guanosine 5'-monophosphate of breast meat decreased as KC grew older. The shear force values of meat from older birds increased concomitantly with an increase in total collagen. Principle component analysis revealed that the meat quality of CB was greatly different from that of KC meat. High shear force values of KC meat at 20 wk of age were well correlated with an increase in the ß-sheet structure (amide I) and amide III of collagen. Raman spectra at 3,207 cm-1 and relative α-helical content were negatively correlated with shear force values of KC breast meat. These could be used as markers to evaluate KC meat quality.

LC-QqQ-MS/MS methodology for determination of purine and pyrimidine derivatives in unifloral honeys and application of chemometrics for their classification.

The new liquid chromatographic-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) method for determination of purine and pyrimidine derivatives in honey produced by Apis mellifera was developed. 13 compounds were determined in total of 130 unifloral honey samples of 13 varieties: uracil, thymine, thymidine, xanthine, guanine, adenine, uridine, pseudouridine, xanthosine, inosine, hypoxanthine, guanosine and cytidine. The levels of some of these compounds varied between the specific honey types. The most abundant were uridine (up to 44.66 mg/kg), xanthine (up to 20.48 mg/kg) and xanthosine (up to 19.22 mg/kg). The data were evaluated by principal component analysis (PCA) and k-nearest neighbors (k-NN) classification (selected 9 and 8 honey types, respectively) to examine differences between the honey varieties allowing their discrimination based on purine and pyrimidine derivatives amounts. The data allowed to distinguish between 8 honey types (balanced accuracy 82%) and for most of the varieties obtained classification rates ranged from 96 to 100%.

DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

The effect of purine content on sensory quality of pork.

Although high level of purine in foods is considered a risk factor for hyperuricemia and gout, purine-rich foods continue to be popular for their delicious taste. The main objective of this study was to investigate the effects of purine bases on the sensory quality of pork. A total of 406 longissimus thoracis et lumborum samples were collected from a heterogeneous F6 pig population to determine purine composition and its correlation to sensory quality of pork. The contents of total purine and two major uricogenic bases (adenine and hypoxanthine) were negatively correlated with tenderness, juiciness, oiliness and overall liking (r < -0.2, P < 0.05), but they were not significantly correlated with umami. In contrast, guanine content, which accounts for only about 10% of the total purine content, was positively correlated with umami (r = 0.15, P < 0.05), and had no significant relationships with other sensory indicators. These results imply that purine bases with different uricogenic effects also influence different sensory quality indices of pork.

The Association between Purine-Rich Food Intake and Hyperuricemia: A Cross-Sectional Study in Chinese Adult Residents.

OBJECTIVE: To explore the correlation between purine-rich food intake and hyperuricemia in Chinese adult residents. METHOD: A cross-sectional study was conducted on the purine-rich food intake of Chinese adult residents based on the China Health and Nutrition Survey (CHNS) in 2009. The subjects were divided into hyperuricemia group and nonhyperuricemia group according to serum uric acid level, and the differences of the sociodemographic information (age, gender, and region), health status (weight status, blood pressure, blood sugar status), living habits (alcohol consumption, smoking status) and food intake (purine-rich food, other food) were compared between the two groups. Logistic regressions investigated the associations between the daily intake of purine-rich food (animal-derived food and legumes) and hyperuricemia. RESULTS: Eventually, 6813 subjects were included in our study, 1111 of them had hyperuricemia. The intake of seafood, legumes, red meat, and poultry all increased the risk of hyperuricemia (p < 0.05), while the intake of purine-rich fungi and purine-rich vegetables did not affect the occurrence of hyperuricemia. Animal-derived food was the main source of purine-rich food consumed by Chinese adult residents (140.67g/day), which had a great impact on hyperuricemia. Finally, after adjusting for gender, age, region, body mass index (BMI), alcohol consumption, hypertension, and refined grains intake, the risk of hyperuricemia increased by 2.40% and 1.10% for each increase of 10 g in animal-derived food intake (OR = 1.024, 95% CI: 1.018-1.030) and legumes intake (OR = 1.011, 95% CI: 1.003-1.019), respectively. CONCLUSION: The intake of animal-derived food and legumes were positively correlated with the occurrence of hyperuricemia. Controlling the intake of animal-derived food and legumes would be more beneficial to controlling the risk of hyperuricemia.

Effect of allicin and its mechanism of action in purine removal in turbot.

Low-purine food is not only the focus of gout patients, but also the focus of contemporary green diet development. Fish are usually considered as high-purine foodstuff because of the high nutritional value and high purine content. Therefore, it is necessary to reduce purine content in fish to ensure that they are suitable for patients with hyperuricemia or gout. In this study, the effect of allicin on purine reduction in turbot during cooking was investigated using high-performance liquid chromatography, and the change of xanthine oxidase (XO) activity was also studied. Molecular docking analysis was further performed to elucidate the mechanism of purine reduction by allicin. The results revealed that in the step of soaking, allicin could reduce purine content in fish by slightly enhancing XO activity, promoting hypoxanthine transformation into xanthine. The removal of total purines in experimental and control group reached 70.45% and 57.20%, respectively. Moreover, allicin could change the thermal stability of xanthine by providing an acidic environment, resulting in the rapid decrease of xanthine and hypoxanthine levels by boiling. Thus, this study provides a simple method to decrease purine levels, suggesting a possibility that allicin can function as a purine remover in food.

Purine molecules in Parkinson's disease: Analytical techniques and clinical implications.

Parkinson's disease (PD) is a neurodegenerative disorder that primarily affects patients over the age of 65. PD is characterized by loss of neurons in the substantia nigra and dopamine deficiency in the striatum. Once PD is clinically diagnosed by the observation of motor dysfunction, the disease is already in its advance stages. Consequently, there is a major push to identify clinical biomarkers that are useful for the earlier detection of PD. Using untargeted metabolomics, several research groups have identified purine molecules, and specifically urate, as important biomarkers related to PD. This review will summarize recent findings in the field of purine metabolomics and biomarker identification for PD, including in the areas of PD pathophysiology, diagnosis, prognosis and treatment. In addition, this article will summarize and examine the primary research techniques that are employed to quantify purine molecules in both experimental systems and human subjects.

Aqueous recognition of purine and pyrimidine bases by an anthracene-based macrocyclic receptor.

A water-soluble bis-anthracenyl tetralactam binds biogenic heterocycles with high affinities in aqueous solution, rising to 107 M-1 for the purine hypoxanthine. Recognition occurs through a combination of hydrogen bonding and hydrophobic interactions, and results in fluorescence changes which suggest applications in sensing.

Enantiospecific electrochemical rearrangement for the synthesis of hindered triazolopyridinone derivatives.

Triazolopyridinone derivatives are of high value in both medicinal and material chemistry. However, the chiral or hindered triazolopyridinone derivatives remain an underexplored area of chemical space because they are difficult to prepare via conventional methods. Here we report an electrochemical rearrangement for the efficient synthesis of otherwise inaccessible triazolopyridinones with diverse alkyl carboxylic acids as starting materials. This enables the efficient preparation of more than 60 functionalized triazolopyridinones under mild conditions in a sustainable manner. This method is evaluated for the late stage modification of bioactive natural products, amino acids and pharmaceuticals, and it is further applied to the decagram scale preparation of enantiopure triazolopyridinones. The control experiments support a mechanism involving an oxidative cyclization and 1,2-carbon migration. This facile and scalable rearrangement demonstrates the power of electrochemical synthesis to access otherwise-inaccessible triazolopyridinones and may find wide application in organic, material and medicinal chemistry.

Gold Nanoparticles as Colorimetric Sensors for the Detection of DNA Bases and Related Compounds.

Results regarding interaction of colloidal gold solutions with nucleobases, including uracil (U), as well as its sulfur derivatives, 2-thiouracil (2TU) and 4-thiouracil (4TU), cytosine (C), adenine (A), and guanine (G), as well as urea and thiourea (TU), are reported. Anionic stabilized citrate gold nanoparticles (AuNPs) were synthesized by reducing the tetrachloroaurate (III) trihydrate with trisodium citrate. The surface plasmon resonance (SPR) band was used in the characterization of synthesized AuNPs, as well as transmission electron microscope (TEM) imaging, which was used in the characterization of dispersed and aggregated gold nanoparticles. Interactions of nucleobases with the gold surface was analyzed by following the plasmon absorbance band red shift of the AuNPs. The sulfur-containing compounds adsorbed to the nanoparticle surfaces by chemisorption-type interactions; with TU and 4TU, the process is accompanied by a sudden change in color; in contrast, 2TU forms stable functionalized gold nanoparticles. Urea and U do not adsorb to nanoparticle surfaces, but the other heterocyclic bases containing nitrogen interact effectively with the gold surface, causing the assembly of nanoparticles, even though the interparticle self-aggregation process was slower than that mediated by either TU or 4TU. The method is efficient in the colorimetric detection of nucleobases and derivatives at concentration levels on the order of 1 µM.

Development of a novel and quick UPLC-MS/MS method for the pharmacokinetic analysis of duvelisib in beagle dogs.

Duvelisib, a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor, was recently approved in the USA as the therapeutic drug for patients with the diseases of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In the present study of our research, a quick and simple bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique was fully explored and established for the quantification of plasma duvelisib concentrations from beagle dog in which gilteritinib was used as the internal standard (IS). After a simple and quick protein precipitation treated with acetonitrile, the chromatographic separation of the analyte was carried out on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) conducted in a gradient elution procedure where acetonitrile (solvent A) and 0.1 % formic acid in water (solvent B) consisted as the mobile phase. The measurements of the analyte and IS were explored using a XEVO TQS triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected reaction monitoring (SRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 416.88 → 281.88 for duvelisib, and m/z 553.09 → 436.01 for IS, respectively. The assay was successfully established in the calibration range from 0.5 to 3000 ng/mL for duvelisib, where the lower limit of quantification (LLOQ) was set at 0.5 ng/mL. The precisions of intra-day and inter-day for duvelisib were all below 12.6 %, and the accuracies were from -2.5% to 14.1%. Both matrix effect and mean recovery of the analyte and IS were all acceptable, and the analyte was stable during the assay and storage in dog plasma samples. The novel established bioanalytical method based on UPLC-MS/MS technique was effectively employed to the investigation of the pharmacokinetic profile of duvelisib in beagle dogs following a 1.34 mg/kg single dose of oral administration.

Time of flight mass spectrometry based in vitro and in vivo metabolite profiling of ribociclib and their toxicity prediction.

The metabolic investigation in the drug discovery process is an imperative aspect for selection of drug candidates with excellent therapeutic efficacy and safety profile. Ribociclib (RIBO), an orally active Cyclin dependent kinases inhibitor recently approved by USFDA for its clinical efficacy against human epithelial growth factor receptor negative and hormonal receptor positive advanced breast cancer. Although an in vitro metabolite identification study of RIBO is available in literature, no systematic metabolic investigation including detailed structural characterization and toxicity prediction of the metabolites generated in in vivo system is reported till date. Therefore, in this study, we focused on the characterization of its entire metabolites generated in in vitro as well as in vivo matrices. In vitro study includes incubation of RIBO in rat and human liver microsomes and human S9 fraction, while in vivo study was carried out using plasma, urine and faeces samples of male Sprague Dawley rats. A total of 22 metabolites were successfully separated on Agilent SB C18 (100 × 4.6 mm, 2.7µ) column using ammonium formate (pH 3.5) and acetonitrile as mobile phase. Metabolites were identified with the help of UHPLC-ESI-Q-TOF-MS/MS by accurate mass measurement. RIBO was found to be metabolised by N- dealkylation, sulphation, acetylation, oxidation, hydroxylation, carbonylation, dehydrogenation and by a combination of these reactions. The in silico toxicity profiling of all the metabolites was carried out with the help of ProTox-II software. Ten out of twenty two newly identified metabolites showed to have potential for possessing immunotoxicity. Novelty of this investigation can be justified by the unavailability of any previously published literature on complete in vitro and in vivo metabolite profiling of RIBO. Moreover, in silico toxicity of the metabolites were also not known till date.

Determination of total purine and purine base content of 80 food products to aid nutritional therapy for gout and hyperuricemia.

The aim of this work is to facilitate the nutritional therapy of gout and hyperuricemia. In Japan, patients with gout or hyperuricemia are recommended to consume less than 400 mg of dietary purines per day. When receiving nutritional therapy for gout or hyperuricemia, purine-rich foods (>200 mg/100 g) should be eaten in even lower quantities. The purine content of foods reported in this study are as follows: noodles, 0.6-12.1 mg/100 g; bread, 4.4 mg/100 g; peas or seeds, 19.6-67.1 mg/100 g; dairy, 0.0-1.4 mg/100 g; Japanese vegetables, 0.9-47.1 mg/100 g; seasonings, 0.7-847.1 mg/100 g; meat or fish, 19.0-385.4 mg/100 g; fish milt, 375.4-559.8 mg/100 g; and supplements, 81.9-516.0 mg/100 g. Foods containing very large amounts of purine (>300 mg/100 g) included anchovy, cutlassfish (hairtail), cod milt, globefish milt, dried Chinese soup stock, dried yeast, a Euglena supplement, and a Lactobacillus supplement. When eating these high-purine food or supplements, the quantity taken at one meal should be limited, especially milt because they typically consumed amount of 20-30 g is equivalent to 75-168 mg total purines. This is 20%-40% of the recommended daily amount (400 mg/day) for patients with gout or hyperuricemia. Thus, these patients should restrict the amount of purine-rich foods they consume. Good dietary habits with a good balance of nutrients are recommended.